Endogenous IFN-alpha production by plasmacytoid dendritic cells exerts an antiviral effect on thymic HIV-1 infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-alpha in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-alpha production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-alpha upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-alpha in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-alpha upon engagement of TLR-9. Importantly, CpG considerably increased IFN-alpha production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-alpha. The degree of inhibition depends on the level of IFN-alpha produced by the thymic pDC.     

Heterocyst patterns without patterning proteins in cyanobacterial filaments

We have quantitatively modeled heterocyst differentiation after fixed nitrogen step-down in the filamentous cyanobacterium Anabaena sp. PCC 7120 without lateral inhibition due to the patterning proteins PatS or HetN. We use cell growth and division together with fixed-nitrogen dynamics and allow heterocysts to differentiate upon the local exhaustion of available fixed nitrogen. Slow transport of fixed nitrogen along a shared periplasmic space allows for fast growing cells to differentiate ahead of their neighbors. Cell-to-cell variability in growth rate determines the initial heterocyst pattern. Early release of fixed nitrogen from committed heterocysts allows a significant fraction of vegetative cells to be retained at later times. We recover the experimental heterocyst spacing distributions and cluster size distributions of Khudyakov and Golden [Khudyakov, I.Y., Golden, J.W., 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040-16045].     

Predicaments of secularism: Muslim identities and politics in Mumbai

Most of the debate about secularism and the secular state in India has remained at a general level, leaving a great many gaps in our knowledge of the actual meanings and practices associated with secularism in India. This article argues that secularism in India is premised on an unstable separation of a realm of politics from a supposedly unpolitical realm of culture, where communities have been represented in rather static and undifferentiated terms. Discussing ethnographic material from Muslim neighbourhoods in Mumbai the author shows how the separation between 'pure' culture and 'dirty' politics is breaking down in the face of a new political assertiveness among ordinary, low-status Muslims. This challenges the position of religious leaders and it also questions widely held assumptions of the relative coherence of the Muslim community.     

Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence

Post-translational modifications (PTMs) occur on almost all proteins analyzed to date. The function of a modified protein is often strongly affected by these modifications and therefore increased knowledge about the potential PTMs of a target protein may increase our understanding of the molecular processes in which it takes part. High-throughput methods for the identification of PTMs are being developed, in particular within the fields of proteomics and mass spectrometry. However, these methods are still in their early stages, and it is indeed advantageous to cut down on the number of experimental steps by integrating computational approaches into the validation procedures. Many advanced methods for the prediction of PTMs exist and many are made publicly available. We describe our experiences with the development of prediction methods for phosphorylation and glycosylation sites and the development of PTM-specific databases. In addition, we discuss novel ideas for PTM visualization (exemplified by kinase landscapes) and improvements for prediction specificity (by using ESS--evolutionary stable sites). As an example, we present a new method for kinase-specific prediction of phosphorylation sites, NetPhosK, which extends our earlier and more general tool, NetPhos. The new server, NetPhosK, is made publicly available at the URL http://www.cbs.dtu.dk/services/NetPhosK/. The issues of underestimation, over-prediction and strategies for improving prediction specificity are also discussed.     

Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Evolution of Multilocus Genetic Structure in an Experimental Barley Population

Data from 311 selfed families isolated from four generations (F8, F13, F23, F45) of an experimental barley population were analyzed to determine patterns of change in character expression for seven quantitative traits, and in single-locus allelic frequencies, and multilocus genetic structure, for 16 Mendelian loci that code for discretely recognizable variants. The analyses showed that large changes in single-locus allelic frequencies and major reorganizations in multilocus genetic structure occurred in each of the generation-to-generation transitions examined. Although associations among a few traits persisted over generations, dynamic dissociations and reassociations occurred among several traits in each generation-transition period. Overall, the restructuring that occurred was characterized by gradual decreases in the number of clusters of associated traits and increases in the number of traits within each cluster. The observed changes in single-locus frequencies and in multilocus genetic structure were attributed to interplay among various evolutionary factors among which natural selection acting in a temporally heterogeneous environment was the guiding force.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

Potamogeton pectinatus Is Constitutively Incapable of Synthesizing Ethylene and Lacks 1-Aminocyclopropane-1-Carboxylic Acid Oxidase

A highly sensitive laser-driven photoacoustic detector responsive to [less than or equal to]2.1 nmol m-3 ethylene (50 parts per trillion [v/v]) was used for ethylene analysis. Dark-grown plants of Potamogeton pectinatus L. growing from small tubers made no ethylene. Exposure of shoots to white light, wounding, submergence in water followed by desubmergence, partial oxygen shortage, indole acetic acid, or carbon dioxide failed to induce ethylene production, although clear effects were observed in Pisum sativum L. Some ethylene was released after applying high concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC; 10 mol m-3) to P. pectinatus, but the amount was trivial compared with that released by P. sativum. More endogenous ACC was found in P. pectinatus than in P. sativum. Considerable ACC oxidase activity was present in tissue extracts of P. sativum. However, no ACC oxidase activity was found in P. pectinatus, indicating that this is where ethylene production is arrested.     

Hepatitis C Viremia Patterns in Incident Hepatitis C Infection and One Year Later in 150 Prospectively Tested Persons Who Inject Drugs

Objectives

To assess HCV viremia levels just before, during and one year after anti-HCV seroconversion in people who inject drugs (PWID).

Methods

PWID enrolling into a needle exchange program in Malmö, Sweden, 1997–2005 constituted the source population. Sera were obtained at enrolment and at approximately 3–4 monthly intervals afterwards, and were initially tested for anti-HIV, HBsAg/anti-HBc and anti-HCV and thereafter for markers previously negative. Seroconversion to anti-HCV had occurred during the study period in 186 out of 332 seronegative subjects. In these anti-HCV seroconverters, quantitative HCV RNA PCR was retrospectively performed on frozen sera to determine viremia levels in the last anti-HCV negative, the first anti-HCV positive and in one year follow-up samples.

Results

Among 150 subjects seroconverting to anti-HCV with samples available from all three defined time-points, eight different patterns of viremia were observed. Spontaneous clearance at one year was noted in 48 cases (32%) and was associated with female gender (p = 0.03, CI 0.17–1.00). In 13 cases HCV-RNA was not detected in any study sample. Among 61 subjects with pre-seroconversion viremia, viral load was significantly higher in the pre-seroconversion samples compared to subsequent samples. For the whole group, viral load declined to undetectable levels at seroconversion in 28% of cases (but with recurrent viremia in 15%).

Conclusions

Different patterns of HCV RNA kinetics were observed among PWID with documented seroconversion to anti-HCV. The frequently observed absence of detectable HCV RNA in the first anti-HCV positive sample (irrespective of subsequent viremia) demonstrates the importance of repeated sampling and RNA testing for determination of the outcome of acute infection.